Commercial production of tomato aims for productivity combined with quality. Quality can be defined in different terms like flavour, taste, texture, mouth feel, appearance, shape, colour, soluble solids, nutritional compounds, disease resistance and shelf-life. During the ripening of fruits these quality traits can develop in various ways depending on the variety in combination with the growing conditions and postharvest treatments. Therefore, the end product, i.e. the fruit that is consumed, often is a compromise between all these traits.
Optimising fruit developmental traits contributes to the profitability of the commercial grower. Plant breeding has traditionally provided the growers with varieties bred for high productivity. Such varieties have been selected to enable the grower to maximise fruit biomass production under specific environmental conditions.
However, recently the fresh market of tomato has changed in the sense that in addition to the traditional varieties, products with improved quality traits like flavour, taste and texture are demanded. This has led to a revision of the breeding targets towards increased quality traits which are preferably combined with high productivity.
A key trait in this respect is shelf-life. Varieties of which harvested fruits can be stored for a longer period of time without losing texture and firmness can be harvested at a later developmental stage. This has the enormous advantage that quality traits can develop during the growth of the crop. In addition, fruits which can ripen without losing texture and firmness can be of interest for the fresh-cut market.
The combined value of the expressed quality traits can differ substantially between commercial varieties. A major obstacle in the improvement of the overall quality of harvested tomato fruits is caused by the fact that the development of quality traits like flavour, colour and taste is often incongruous with the desire to harvest fruits with a long shelf-life. A long shelf-life is required in order to avoid too much bruising during harvest and storage. As fruit ripening in terms of colouration and softening continues postharvest, a solution to this problem is often found by harvesting the tomato fruits at the mature green or breaker stage after which they will turn red during storage. The big advantage of such practice is that the fruits are still very firm at harvest and therefore have a high resistance against bruising. The fruits will reach the consumer red-coloured and undamaged. Although this is a practical solution to the shelf-life limitation, in cases where the products need to be stored for prolonged periods of time e.g. when long transportation distances are involved this approach is still inadequate.
A further and very important problem is that although colouration and softening develop postharvest, flavour and taste do not. Therefore the quality trait shelf-life seems to be in conflict with the quality traits flavour and taste. It is therefore desirable to improve tomatoes in such a way that quality traits like flavour and taste can develop preharvest in combination with a long shelf-life.
A further advantage of long shelf-life in tomato is related to the labour input required to harvest the fruits. Fruits with a normal shelf-life need to be picked as much as possible at the same developmental stage in order to prevent too much variation with respect to the post-harvest quality of the fruit due to variation in maturity. This can sometimes even be twice a day. In case long shelf-life tomatoes are available there is no need for such labour intensive harvesting, as irrespective of the developmental stage at harvest fruits will ripen and remain firm. In addition to the reduced labour input, flexibility in harvesting time allows to tailor the delivery of the produce to the market demand.
As ethylene is a strong stimulator of ripening, previous attempts to improve shelf-life of tomato fruits involve selecting genetic variants with fruits which either produce less ethylene or are less sensitive to ethylene. This has resulted in the identification of a number of pleiotropic ripening mutants with improved shelf-life which have been characterised to different levels of detail (Giovannoni, J (2007) Current Opinion in Plant Biology 10, 283-289). For example the Never-ripe (NR) mutant has been shown to be mutated in an ethylene receptor gene which resulted in insensitvity to ethylene. Due to this mutation the fruits remain firm during postharvest storage but ripening and the associated development of colour and taste is blocked.
In addition, ripening-inhibitor (rin), non-ripening (nor) and colourless non-ripening (cnr) mutants have been identified which are modified in genes encoding transcription factors involved in the production of, or response to, ethylene.
Although mutants like rin have a certain practical value for a better shelf-life, there is still room for improvement. In a preferred situation increased shelf-life should be achieved without compromising positive ripening-associated quality traits like pigmentation, flavour, and texture.
For tomato fruit growth and development a number of consecutive phases can be discerned. The earliest phase is floral development. After pollination as a second phase, early fruit development takes place which is characterised by a high frequency of cell division. During the third phase, the fruit is rapidly increasing in size mainly due to cell expansion. At the end of the third phase the fruit reaches the mature green stage. During the fourth phase fruit ripening takes place which is characterised by a change in colour and flavour as well as fruit firmness and texture.
The build up of the characteristic red colour of the tomato fruit is caused by the accumulation of lycopene and carotene. In general, different colouration phases are distinguished: mature green, breaker, pink and red. The typical red pigmentation initiates at the breaker stage. Red ripe stage or red ripe harvested fruit stage is the stage where the fruit has reached its mature colour on the major part of the fruit. In addition, enzymatic activity leads to degradation of the middle lamellar region of the cell walls which leads to cell loosening which is manifested as softening and loss of texture of the fruit. Softening of the fruit is often measured as external resistance to compression which can be quantified for example by a penetrometer.
Detailed molecular and biochemical studies have shown activities like endo-polygalacturonase and pectin-methyl-esterase to be involved in fruit softening. Antisense inhibition of the genes encoding these enzymes generally did not result in an improvement of fruit firmness which demonstrates that other activities are involved in the overall softening process. In this respect, expansins related to fruit ripening have been identified as being involved in the fruit softening process. Antisense inhibition of a ripening associated expansin indeed resulted in a small reduction in the rate of fruit softening.
As an alternative approach to increase shelf-life of tomato fruits, deoxyhypusine synthase (DHS) was suppressed transgenically (Wang, T. et al (2005) Plant Physiology 138, 1372-1382). Fruits of transgenic plants showed normal ripening in terms of colouration but a reduction in postharvest softening and senescence related to the level of DHS suppression. Some of the events were free of wrinkling of the fruit skin for up to 44 days after harvest of the fruit at the breaker stage. However, strongly suppressed DHS events showed pleiotropic effects such as male sterility probably due to the fact that DHS modulates several translation initiation factors 5A (eIF-5A).
In addition, a naturally occurring mutation has been described called Delayed Fruit Deterioration (DFD) which is characterised by a very long shelf-life of up to 7 months (Saladie, M. et al (2007) Plant Physiology 144, 1012-1028). This mutant has a high resistance to external compression of the fruit and minimal water loss but internal tissues undergo a normal softening. This demonstrates that softening of fruit tissue and fruit firmness are not necessarily linked.
The conclusion of these studies is that probably different physiological processes are involved in the overall fruit softening process. Modification of single genes known to be involved in ripening has not yet resulted in a fruit with normal ripening but minimal tissue softening. The conclusion could be that it is physiologically not feasible to modify ripening this way.
Alternatively, as many genetic factors are involved in the ripening process it may be required to modify these genes simultaneously or the critical factor has not yet been identified.
As tomato is a climacteric fruit, the ripening phase is characterised by an enhanced ethylene production and respiratory burst. Respiration is the metabolic oxidation of sugars which leads to the release of CO2. As a by-product of this respiratory activity reactive oxygen species (ROS) are formed which are very reactive and can cause significant damage to cell structures leading to oxidative stress. ROS are suggested to play an important role in the enhancement of senescence in both leaves and fruits. During the climacteric phase flavour (volatiles, sugars, acids) and colour compounds are formed which provide a tomato fruit its typical taste perception and appearance.
The senescence phase is the final ripening phase which is characterised by a further softening of the fruit tissue, increased respiration and water loss which further facilitates seed dispersal. Infection by opportunistic pathogens like Botrytis may occur relatively easy at this stage.
As tomato is climacteric, fruit can be picked at the mature green or breaker/pink stage after which the colouration and softening processes continue to take place postharvest. If required, the harvested immature fruits can be exposed to exogenous ethylene in order to enhance the ripening process. Given the important stimulating role of ethylene in the ripening process, efforts to increase shelf-life have focused on the ethylene biosynthesis, perception or effector genes in order to slow down fruit ripening. Both through selection of natural variation as well as through genetic engineering ethylene components have been modified successfully which has resulted in extended shelf-life through slowing down the ripening process. The down side of such approach is that desirable quality traits related to fruit ripening develop more slowly as well.
Senescence is a naturally occurring, developmental process at the end of a life cycle of a plant or plant organ like a leaf or a fruit. Well-known stimulating factors of senescence are developmental age, wounding, detachment, darkness, nutrient deficiency and hormones. Although ethylene is the plant hormone known to stimulate senescence other hormones like jasmonate may also contribute to this process. During the final stage of leaf development metabolism is reprogrammed in order to remobilize resources into reproductive structures like seeds.
Yellowing of leaves being the most visible symptom of senescence is a consequence of chlorophyll breakdown during a relatively late stage of senescence which can be enhanced by ethylene once a leaf is receptive. Senescence is also considered the terminal stage of fruit ripening. The process is characterised by extensive tissue softening, water loss and deterioration which can serve seed dispersal. In addition to ethylene biosynthesis and response, postharvest metabolism of detached fruit is characterised by a strong enhancement of respiration which as a consequence leads to the production of reactive oxygen species (ROS).
Oxidative stress is known to contribute significantly to senescence but as compared to ethylene has not been studied extensively for fruit ripening. One study describes a correlation of fruit deterioration and the level of ROS scavenging enzymes which at least suggests a functional role for these enzymes in fruit senescence (Mondal, K. et al (2004) Biologia Plantarum 48, 49-53).
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